Fig. 4 | Laboratory Investigation

Fig. 4

From: The Shh/Gli signaling cascade regulates myofibroblastic activation of lung-resident mesenchymal stem cells via the modulation of Wnt10a expression during pulmonary fibrogenesis

Fig. 4

The sonic hedgehog pathway regulates pulmonary fibrotic events via Wnt/β-catenin signaling. a–c Mice (n = 6 per group) were intraperitoneally injected with vehicle (10% DMSO/saline) or 25 mg/kg Gant61 three times weekly as indicated 7 days after bleomycin (BLM) administration. Mice were killed on day 14 after BLM instillation. a Collagen deposition in mouse lung tissues was revealed by Sirius red staining. b The mRNA levels of fibroblast-specific protein-1 (FSP-1) and fibronectin in mouse lung tissues were determined by qRT-PCR. The results are shown as the means ± SD (*P < 0.05 versus BLM + vehicle). c The protein levels of α-smooth muscle actin (α-SMA), collagen I, fibronectin, and stem cell antigen-1 (Sca-1) in mouse lung tissues were measured by western blot. d Mouse lung-resident mesenchymal stem cells (LR-MSCs) were pretreated with 5 μM Gant61 for 1 h followed by stimulation with 10 ng/ml transforming growth factor-β1 (TGF-β1) for 48 h. The expression of α-SMA and collagen I in mouse LR-MSCs was examined by immunofluorescence. Mouse LR-MSCs were incubated with 100 ng/ml recombinant sonic hedgehog (Shh) for various durations as indicated, and the expression of Wnt10a (e) and nuclear translocation of p-β-catenin and β-catenin (f) were analyzed by western blot. Lamin B was used as the nuclear marker. g Mice were treated as described in a–c. Wnt10a expression in mouse lung tissues was analyzed by immunohistochemistry. h–j Mouse LR-MSCs were treated as described in d. h The protein levels of Wnt10a in mouse LR-MSCs were measured by western blot. i Cytoplasmic and nuclear expression of glioblastoma1 (Gli1), Gli2, p-β-catenin, and β-catenin in mouse LR-MSCs were determined by western blot. Lamin B and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as the loading controls for nuclear and cytoplasmic proteins, respectively. j The association of Gli1 with the Wnt10a promoter region in mouse LR-MSCs was analyzed by chromatin immunoprecipitation (ChIP). Normal mouse IgG was used as the control antibody. A representative gel image is shown, and the expression levels were quantified by densitometry and normalized to the input. The results are shown as the means ± SD (*P < 0.05 versus TGF-β1)

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